Lipids are an apparently essential requirement for most animal cell culture. [See Yamane et al., Proc. Japan Acad. 57B(10):385-389 at p. 388 (1981); and Iscove, "Culture of Lymphocytes and Hemopoietic Cells in Serum-Free Medium," In Barnes et al. (eds.), Methods for Serum-Free Culture of Neuronal and Lymphoid Cells, pp. 169-185 (1984), where it is stated at page 170: "If the experience with lymphocytes and hemopoietic cells can be extrapolated, lipids may turn out to be a universal requirement for freshly explanted cells."] Specific lipid requirements have been identified for mammalian cell lines (CHO) useful for recombinant protein expressing [Ham, Proc. Natl. Proc. Nat. Acad. Sci., 1965 53:288], for insect cell lines (Sf9 [Wilke, et al., 1980, Develop. Biol. Standard, 46:29-37] and for antibody secreting cell lines [Kawamoto et al., 1983 Analytical Biochem. 130:445-453]. The direct addition of lipids to media is not practical due to their low solubility. Conventionally, in serum-containing media, lipids are provided to the medium in the serum wherein the lipids are carried as water soluble lipoproteins; whereas conventionally, in serum-free media, lipids are carried by albumin to which the lipid is coupled through a process employing a solvent, such as chloroform. [Goodwin et al., In Kurstak et al., (eds.), Invertebrate Systems In Vitro, pp. 493-508 (1980); Iscove, id.].
Although conventional culture media have been generally prepared by adding serum in large amounts (about 5 to 20%), the use of serum has a number of disadvantages including (1) expense, in that, more than half of the cost of the media is usually occupied by the costs of the sera; (2) time necessary to check quality of sera which may vary from lot to lot and can be contaminated with mycoplasma or virus; and (3) downstream purification problems, in that, sera can contain various unidentified proteins. Similar considerations pertain to the use of albumin, a major plasma protein in conventional serum-free media. [See Yamane et al., Proc. Japan Acad., 57B(10):385-389 (1981) wherein it is stated at page 388: "BSA is, needless to say, a serum protein which is often discrepant in its cell growth promoting action of each commercial preparation."] As it is difficult to purify albumin on a large scale, commercially available albumin preparations can contain other contamination proteins, and not even crystallized preparations exceed 98% purity. "In addition to contaminating proteins, albumin preparations can be presumed to contain other hydrophobic substances including fatty acids and steroid hormones, and traces of salts and buffers used in the purification along with their contaminating transition metal ions." Iscove, id. at page 176. Therefore, it would be advantageous to eliminate from cell culture media both serum and albumin, as well as other proteins as lipid carriers. This invention provides the option of eliminating such lipid carriers.
Attempts have been described in the literature to supply lipids as liposomes to cells in culture [N. N. Iscove, Culture of Lymphocytes and Hemopoietic Cells in Serum-Free Medium, p. 169-185, in D. W. Barnes, D. A. Sibasku, G. H. Sato (eds.), Methods for Serum-Free Culture of Neuronal and Lymphoid Cells, Alan R. Liss, NY (1984)], Poste, G. D. et al., Methods in Cell Biology, 14:43-71 (1976), D. M. Prescott, (ed.)], and M. Kriegler, Cell, 33(2):413-422 (1983). These liposomes are most typically prepared by sonicating the lipid mix in the presence of a protein (albumin) and result in particles of size typically about 25 nm [H. J. Morton; How to Prepare Media Containing Fat-Soluble Substances, Chapter 23, p. 353-357, in C. Waymouth, R. G. Ham, P. J. Chapple (eds.), The Growth Requirements of Vertebrate Cells In Vitro, Cambridge University Press, Cambridge, 1981.
Iscove, id. at page 179-181, provides an example of the prior art process used to supply lipids to serum-free culture. In such a process, lipids are mixed by being dissolved in chloroform; the chloroform is then evaporated and the lipids are "dried onto the bottom of the beaker." A suspending medium containing albumin is then added and the mixture is sonicated to disperse the lipids in the form of vesicles--liposomes small enough to pass through the pores of a sterilizing filter (0.45 .mu.m). Iscove's following description of such a process points out some of the inherent problems of supplying lipids to culture medium in such a manner:
At the end of the sonication period, a few small aggregates of soybean lipid may have escaped dispersion. These are ignored. The suspension is passed once through a membrane of 1.2 .mu.m pore size . . . after which it usually passes without difficulty through a sterilizing 0.45 .mu.m filter. If the filter blocks rapidly, it may be helpful to pass the suspension once more through a 1.2 .mu.m membrane. The grey residue on the filter is particulate metal shredded from the sonicator probe. Filtration is easiest immediately after sonication and should not be delayed . . . . PA0 The resulting suspensions are stored at 4.degree. C. They may become somewhat more opaque in the first day or two of storage. This change probably reflects some coalescence of the liposomes to larger average size but has no influence on their effectiveness in culture . . . . PA0 Because of quantitative variation in the completeness of dispersion and retention on the sterilizing filters, each individual lipid preparation should be titrated in culture to determine its optimal concentration . . . . PA0 Filter-sterilization of medium which has already been supplemented with lipid should be avoided, since there is a risk of retaining an indeterminate amount of lipid on the filter. [Emphasis added.]
The introduction of lipids to cell culture media according to this invention enhances the availability of lipids to the cells and avoids globular lipid aggregates that can be lost in filtration and thus become unavailable to the cells. This invention further provides the option of not having to filter sterilize the lipid component and the rest of the media components separately.